Composition of matter and process

ABSTRACT

Novel antibiotic U-68,204 is produced in a fermentation under controlled conditions using a biologically pure culture of the microorganism Streptomyces thiolactonus, NRRL 15439. This antibiotic is active against various Gram-positive bacteria. Thus, antibiotic U-68,204 can be used in various environments to eradicate or control such bacteria.

DESCRIPTION BACKGROUND OF THE INVENTION

Antibiotic U-68,204, of the subject application, is related tothiolactomycin. See H. Sasaki, et al., J. Antibiotics, 35, 396 (1982).It also is related to citreothiolactone, but not as closely. See Y.Shizuri, et al., Tet. Letters, 24, 1053 (1983). Further, U-68,204 isrelated to thiotetromycin. See S. Omura, et al., J. Antibiotics, 36, 109(1983). Antibiotic U-68,204 differs structurally from thiotetromycin inthat one of the methyl groups of thiotetromycin is converted to anamide. The structural similarities are as shown in Chart I.

BRIEF SUMMARY OF THE INVENTION

Antibiotic U-68,204 is producible in a fermentation under controlledconditions by a biologically pure culture of the new microorganismStreptomyces thiolactonus, NRRL 15439.

Antibiotic U-68,204 is an acidic compound which is active againstvarious Gram-positive bacteria. Thus, antibiotic U-68,204 can be used todisinfect washed and stacked food utensils contaminated with S. aureus.It can also be used as disinfectant on various dental and medicalequipment contaminated with S. aureus. Still further, antibioticU-68,204 can be used as a bacteriostatic rinse for laundered clothes andfor impregnating papers and fabrics; it is also useful for suppressingthe growth of sensitive organisms in plate assays and othermicrobiological media.

DETAILED DESCRIPTION OF THE INVENTION

Chemical and Physical Properties of Antibiotic U-68,204:

Molecular Weight: 267.

Elemental Analysis: Found: C, 58.32; H, 6.68; N, 5.01; S, 10.91 Calc'd:C, 58.43; H, 6.37; N, 5.24; S, 11.99.

Color of Crystals: White.

Molecular Formula: C₁₃ H₁₇ NO₃ S.

Melting Point: 98°-101°.

Infrared Absorption Spectrum:

Antibiotic U-68,204 has a characteristic infrared absorption spectrumusing the attenuated internal reflectance technique as shown in FIG. 1of the drawings. Peaks are observed at the following wavelengths.

    ______________________________________                                                Band                                                                          Freq..sup.1                                                                         Inten..sup.2                                                    ______________________________________                                                3350  W                                                                       3210  W                                                                       2970  W                                                                       2930  W                                                                       1610  S                                                                       1575  sh                                                                      1460  W                                                                       1410  W                                                                       1365  W                                                                       1340  W                                                                       1300  M                                                                        990  W                                                                        910  W                                                                        845  W                                                               ______________________________________                                         .sup.1 Wave numbers (cm.sup.-1)                                               .sup.2 S = Strong                                                             M = Medium                                                                    W = Weak                                                                      sh = shoulder                                                            

UV Spectrum:

The UV spectrum at pH 10 for a 0.0125 mg/ml solution gave maxima at A₃₀₃=0.6199 (ε=13,200) and A₂₃₈ =1.452 (ε=31,025). At pH 2 there was asingle maximum with A₂₃₉ =1.489 (ε=31,800). The UV spectra are shown inFIG. 2.

¹ H-Nuclear Magnetic Resonance (NMR) Spectrum:

The ¹ H-NMR spectrum of antibiotic U-68,204 is shown in FIG. 3 of thedrawings. The ¹ H-NMR spectrum was recorded on a Varian CFT-80Spectrometer in a solution (ca. 0.3 ml., ca. 150 mg/ml) of the sample ofthe antibiotic in deutero chloroform (CDCl₃). The spectrum wascalibrated against tetramethylsilane and frequencies were recorded inppm downfield from tetramethylsilane.

¹³ C-NMR Spectrum:

The spectra were recorded on a CFT-80 spectrophotometer and are shown inFIG. 4. Crystalline samples of the free acid and of the ammonium saltgave the following results:

    ______________________________________                                               The Acid      The Salt                                                 ______________________________________                                               195δ, S 196δ, S                                                   182, S        191, S                                                          175, S        174, S                                                          143, D        144, D                                                          140, S        137, D                                                          132, D        136, S                                                          116, S        112, T                                                          115, T        105, S                                                           56, S         59, S                                                           47, T         49, T                                                           17, Q         18, Q                                                           14, T         15, T                                                           13, Q         13, Q                                                   ______________________________________                                         δ = ppm from internal TMS                                               S = singlet                                                                   D = doublet                                                                   T = triplet                                                                   Q = quartet                                                              

Optical Rotation: [α]_(D) ²⁵ =+177° (c=0.8925, ethanol).

Solubilities:

Antibiotic U-68,204 is soluble in most organic solvents; it is notsoluble in water.

Antimicrobial Spectrum of Antibiotic U-68,204:

Antibiotic U-68,204 is active against various Gram-positive bacteria asshown in the following table.

    ______________________________________                                        MINIMUM INHIBITORY CONCENTRATION                                              Organism Name     Culture UC #                                                                              mcg/ml                                          ______________________________________                                        Staphylococcus aureus                                                                            76           75                                            Staphylococcus aureus                                                                           6675          75                                            Staphylococcus aureus                                                                           3665          75                                            Staphylococcus aureus                                                                           6685         600                                            Streptococcus pyogenes                                                                           152        >600                                            Streptococcus pneumoniae                                                                         41         >600                                            Streptococcus faecalis                                                                           694        >600                                            Escherichia coli   311         600                                            Klebsiella pneumoniae                                                                            58          300                                            Enterobacter cloacae                                                                            9382        >600                                            Pseudomonas aeruginosa                                                                          6676        >600                                            Pseudomonas aeruginosa                                                                          6432        >600                                            Pseudomonas aeruginosa                                                                          6954        >600                                            Pseudomonas aeruginosa                                                                          6955          19                                            Pseudomonas aeruginosa                                                                          9027          37.5                                          Serratia marcescens                                                                             6888        >600                                            Citrobacter freundii                                                                            3507        >600                                            Haemophilus influenzae                                                                          6482          9.4                                           Haemophilus influenzae                                                                          6483          19                                            ______________________________________                                    

The assay is a standard tube dilution assay using BHI (Brain HeartInfusion medium-Difco). "UC" is a registered trademark of The UpjohnCompany Culture Collection.

DESCRIPTION OF THE DRAWINGS

FIG. 1--Infrared absorption spectrum of antibiotic U-68,204.

FIG. 2--UV spectrum of U-68,204.

FIG. 3--¹ -Nuclear Magnetic Resonance (NMR) spectrum of antibioticU-68,204.

FIG. 4--¹³ C-NMR spectrum of U-68,204.

The structure of Antibiotic U-68,204, along with the assignments of theCMR and PMR chemical shifts, is shown in Chart II.

THE MICROORGANISM

The microorganism used for the production of antibiotic U-68,204 is abiologically pure culture of Streptomyces thiolactonus NRRL 15439.

A subculture of this microorganism can be obtained from the permanentcollection of the Northern Regional Research Laboratory, U.S. Departmentof Agriculture, Peoria, Ill., United States. A viable subculture wasdeposited on May 25, 1983, and its accession number in this depositoryis NRRL 15439. It should be understood that the availability of theculture does not constitute a license to practice the invention inderogation of patent rights granted with the subject instrument bygovernmental action.

The microorganism of this invention was studied and characterized byAlma Dietz of the Upjohn Research Laboratories.

Streptomyces thiolactonus NRRL 15439 Color Characteristics

Aerial mycelium predominantly blue. Melanin-positive. The color patternon Ektachrome is given in Table 1. Reference color characteristics aregiven in Table 2. The culture may be placed in the Blue (B) color seriesof Tresner and Backus [Tresner, H. D., and E. J. Backus. 1963. System ofcolor wheels for streptomycete taxonomy. Appl. Microbiol. 11:335-338].

Microscopic Characteristics

Spores are in chains that appear to be tight spirals when observed withthe phase contrast microscope. The chains appear loosely coiled whenobserved with the scanning electron microscope. The spores are sphericalto slightly elliptical and are covered with short spines and fine hairs.The spores measure 0.74×0.65 μm in size.

Growth on Carbon Compounds

The synthetic medium of Shirling and Gottlieb [Shirling, E. B., and D.Gottlieb. 1966] and methods for characterization of Streptomyces species[Int. J. Syst. Bacteriol. 16:313-340] were used for this determination.Growth was good on the positive control (D-glucose), L-arabinose,sucrose, D-xylose, D-mannitol, D-fructose, rhamnose, and raffinose.There was no growth on cellulose. Growth on the negative control((synthetic medium (ISP-9) without added carbon compound)) was doubtful.

Whole Cell Analysis

L-Diaminopimelic acid was detected in whole-cell hydrolysates.

Culture Characteristics

General characteristics are given in Table 3.

Temperature

Growth on Bennett's, Czapek's sucrose, and maltose-tryptone agars wasfair at 18° C., good at 24°-45° C., and poor at 55° C.

Streptomyces thiolactonus NRRL 15439 is readily distinguishedmacroscopically by its blue aerial growth and red-brown-to-brown reversecolors on many media used for characterizing Streptomyces species.Cultures with blue aerial growth have been the subject of a number ofpublications (the Caeruleus series of Baldacci [Baldacci, E. 1959.Extension of the classification of the actinomycetes. Mikrobiologiya(Trans.) 28:258-267], the Coerulescens series of Gauze [Gauze, G. F., T.P. Preobrazhenskaya, E. S. Kudrina, N. O. Blinov, I. D. Ryabova, and M.A. Iveshnikova. 1957. Problems in the classification of antagonisticactinomycetes. State Publishing House for Medical Literature, Moscow.English edition translated by Fritz Danga; David Gottlieb (ed.). TheAmerican Institute of Biological Sciences, Washington, D.C.], theprasinus or azureus-glaucus grouping of Hutter [Hutter, R. 1967.Systematik der Streptomycetes unter besonderer Berucksichtigung der vonihnen gebildeten Antibiotica. S. Karger, Basel], and theviridochromogenes (blue spore) series of Trejo and Bennett [Trejo, W. H.and R. E. Bennett. 1963. Streptomyces species comprising the blue-sporeseries. J. Bacteriol. 85:676-690]. Grouping of cultures with blue aerialgrowth is found in Kuster [Kuster, E. 1972. Simple working key for theclassification and identification of named taxa included in theInternational Streptomyces Project. Int. J. Syst. Bacteriol. 22:139-148]and in Tables 17.45b-e in Family VII. Streptomycetaceae Waksman andHenrici 1943, by Pridham and Tresner in Bergey's Manual, 8thed.[Pridham, T. G., and H. D. Tresner. 1974. Part 17. Actinomycetes andrelated organisms. Family VII. Streptomycetaceae Waksman and Henrici1943. Genus I. Streptomyces Waksman and Henrici 1943. Pages 819-824 inBuchanan and Gibbons, eds., Bergey's Manual of DeterminativeBacteriology, 8th ed. The Williams and Wilkins Co., Baltimore]. Mostcultures with blue aerial growth are melanin-positive, grow well oncarbon compounds in the synthetic medium of Shirling and Gottlieb[Shirling, E. B., and D. Gottlieb. 1966. Methods for characterization ofStreptomyces species. Int. J. Syst. Bacteriol. 16:313-340], arethermoduric, and are noted for the production of distinctly differentantibiotics. These properties can be determined by reading thedescriptions of the blue cultures on pages 819-824 of Bergey's Manual,8th ed. [Pridham, T. G., and H. D. Tresner. 1974. Part 17. Actinomycetesand related organisms. Family VII. Streptomycetaceae Waksman and Henrici1943. Genus I. Streptomyces Waksman and Henrici 1943. Pages 819-824 inBuchanan and Gibbons, eds., Bergey's Manual of DeterminativeBacteriology, 8th ed. The Williams and Wilkins Co., Baltimore] and theoriginal citations given with the descriptions.

The culture characterized in this report differs from those in thereferences cited in its macro- and micro-morphology, its generalcultural characteristics, and in its production of the new antibioticU-68,204 (a new thiolactone). It is proposed that this new culture bedesignated Streptomyces thiolactonus Dietz sp. n. It is understood that,in accordance with the Rules of Nomenclature of Bacteria [Lapage, S. P.,P. H. A. Sneath, E. F. Lessel, V. B. D. Skerman, H. P. R. Seeliger, andW. A. Clark, ed. 1975. International Code of Nomenclature of Bacteria,1976 Revision. American Society for Microbiology, Washington, D.C.],this is the type strain and that, should another strain be found, thetype strain would also be the type subspecies.

                  TABLE 1                                                         ______________________________________                                        Color Characteristics* on Ektachrome [Dietz, A. 1954.                         Ektachrome transparencies as aids in actinomycete classification.             Ann. N.Y. Acad. Sci. 60:152-154]                                                          Streptomyces thiolactonus NRRL 15439                              Agar Medium   Chip     Color                                                  ______________________________________                                        Bennett's  S      185      pale blue                                                     R       59      dark brown                                         Czapek's sucrose                                                                         S      190      light bluish gray                                             R       59      dark brown                                         Maltose-tryptone                                                                         S      190      light bluish gray                                             R       59      dark brown                                         Peptone-iron                                                                             S      159      dark brown                                                    R       59      dark brown                                         0.1% Tyrosine                                                                            S      190      pale bluish gray                                              R       43      moderate reddish brown                             Casein starch                                                                            S      190      pale bluish gray                                              R       77      moderate yellowish brown                           ______________________________________                                         S = surface                                                                   R = reverse                                                                   *Growth on media in tubes was photographed after seven days incubation at     28 C. Color was determined by comparison with NBS color chips [SP 440.        Color: Universal Language and Dictionary of Names. U.S. Government            Printing Office, Washington D.C. 2040 2]; [SRM 2106. ISCCNBS Centroid         Color Charts. Office of Standard Reference Material, Room B311, Chem.         Building, National Bureau of Standards, Washington, D.C. 20234].         

                  TABLE 2                                                         ______________________________________                                        Reference Color Characteristics*                                              Agar          Streptomyces thiolactonus NRRL 15439                            Medium        Chip     Color                                                  ______________________________________                                        Bennett's  S      190      light bluish gray                                             R      44       dark reddish brown                                            P      57       light brown                                        Czapek's sucrose                                                                         S      189      bluish white                                                  R      77       strong yellowish brown                                        P      84       strong yellow                                      Maltose-tryptone                                                                         S      190      light bluish gray                                             R      59       dark brown                                                    P      57       light brown                                        Yeast extract-                                                                           S      190      light bluish gray                                  malt extract                                                                             R      59       dark brown                                         (ISP-2)    P      57       light brown                                        Oatmeal    S      190      light bluish brown                                 (ISP-3)    R      57       light brown                                                   P      76       light yellowish brown                                                         (around growth)                                    Inorganic salts                                                                          S      90       light bluish gray                                  starch     R      91       dark grayish yellow                                (ISP-4)    P      32       brownish pink                                                                 (around growth)                                    Glycerol-  S      190      light bluish gray                                  asparagine R      44       dark reddish brown                                 (ISP-5)    P      58       moderate reddish brown                             ______________________________________                                         S = surface                                                                   R = reverse                                                                   P = pigment                                                                   *Color determination was made on growth on plates incubated 14 days at 28     C. Color was determined by comparison with NBS color chips.              

                  TABLE 3                                                         ______________________________________                                        Culture Characteristics - General                                             Medium                                                                        Agar          Streptomyces thiolactonus NRRL 15439                            ______________________________________                                        Peptone-iron                                                                             S      pale blue-white                                                        R      brown                                                                  P      brown                                                                  O      melanin positive                                            Calcium malate                                                                           S      pale blue-white                                                        R      pale yellow                                                            P      none                                                                   O      malate not solubilized                                      Glucose-   S      trace blue-white                                            asparagine R      pink-tan                                                               P      pink-tan                                                    Skim milk  S      very slight trace blue-white                                           R      maroon                                                                 P      maroon around growth                                                   O      casein not solubilized                                      Tyrosine   S      very pale blue-white                                                   R      reddish brown                                                          P      reddish brown                                                          O      tyrosine solubilized                                        Xanthine   S      very pale blue-white                                                   R      dull yellow-cream                                                      P      pale yellow                                                            O      xanthine solubilized                                        Nutrient starch                                                                          S      pale blue                                                              R      pale olive                                                             P      pale yellow                                                            O      starch solubilized                                          Yeast extract-                                                                           S      pale blue-white                                             malt extract                                                                             R      red-orange                                                             P      red-orange                                                  Peptone-   S      brown                                                       yeast extract-                                                                           R      brown                                                       iron       P      brown                                                       (ISP-6)    O      melanin positive                                            Tyrosine   S      light tan                                                   (ISP-7)    R      red-brown                                                              P      red-brown                                                              O      melanin positive                                            Gelatin                                                                       Plain      S      brown ring                                                             P      brown 1/5, tan-brown 4/5                                               O      trace liquefaction in brown pig-                                              ment area (one of three)                                    Nutrient   S      brown ring                                                             P      brown 1/5, tan-brown 4/5                                               O      trace liquefaction in brown pig-                                              ment area (one of three)                                    Broth                                                                         Synthetic nitrate                                                                        S      pale yellow pellicle and ring                                          P      yellow-tan diffusing from pellicle                                     O      nitrate reduction: + in one, - in                                             two                                                                           trace botton growth                                         Nutrient nitrate                                                                         S      trace blue aerial growth on surface                                           pellicle                                                               P      brown                                                                  O      nitrate reduction: - in all                                                   red with zinc dust                                          Litmus milk                                                                              S      red-tan ring                                                           P      none                                                                   O      slight reduction and peptonization at                                         pH 5.86.                                                    ______________________________________                                         S = surface                                                                   R = reverse                                                                   P = pigment                                                                   O = other characteristics                                                

The compound of the invention process is produced when the elaboratingorganism is grown in an aqueous nutrient medium under submerged aerobicconditions. It is to be understood, also, that for its preparationsurface cultures and bottles can be employed. The organism is grown in anutrient medium containing a carbon source, for example, an assimilablecarbohydrate, and a nitrogen source, for example, an assimilablenitrogen compound or proteinaceous material. Preferred carbon sourcesinclude glucose, brown sugar, sucrose, glycerol, starch, cornstarch,lactose, dextrin, molasses, and the like. Preferred nitrogen sourcesinclude cornsteep liquor, yeast, autolyzed brewer's yeast with milksolids, soybean meal, cottonseed meal, cornmeal, milk solids, pancreaticdigest of casein, fish meal, distillers' dried solids, animal peptoneliquors, meat and bone scraps, and the like. Combinations of thesecarbon and nitrogen sources can be used advantageously.

Production of the compound by the invention process can be effected atany temperature conducive to satisfactory growth of the microorganism,for example, between about 18° and 40° C., and preferably between about20° and 28° C. Ordinarily, optimum production of the compound isobtained in about 2 to 15 days. The medium normally remains alkalineduring the fermentation. The final pH is dependent, in part, on thebuffers present, if any, and in part on the initial pH of the culturemedium.

When growth is carried out in large vessls and tanks, it is preferableto use the vegetative form, rather than the spore form, of themicroorganism for inoculation to avoid a pronounced lag in theproduction of the compound and the attendant inefficient utilization ofthe equipment. Accordingly, it is desirable to produce a vegetativeinoculum in a nutrient broth culture by inoculating this broth culturewith an aliquot from a soil, agar plug stored in liquid N₂, or a slantculture. When a young, active vegetative inoculum has thus been secured,it is transferred aseptically to large vessels or tanks. The medium inwhich the vegetative inoculum is produced can be the same as, ordifferent from, that utilized for the production of the compound, solong as a good growth of the microorganism is obtained.

A variety of procedures can be employed in the isolation andpurification of the compound produced by the subject invention fromfermentation beers. Isolation can be accomplished by solvent extraction,and adsorption on non-ionic macroporous resins. Chromatography on silicagel can be used to purify crude preparations of the antibiotic.

In a preferred recovery process, the compound produced by the subjectprocess is recovered from the culture medium by separation of themycelia and undissolved solids by conventional means, such as byfiltration or centrifugation, and resin adsorption of the filteredbroth. The antibiotic of the subject invention can be recovered from thefiltered beer by resin sorption on a resin comprising a non-ionicmacroporous copolymer of styrene cross-linked with divinylbenzene.Suitable resins are Amberlite XAD-2 and XAD-4, according to theprocedure disclosed in U.S. Pat. No. 3,515,717. (Amberlite resins areavailable from Rohm and Haas, Philadelphia, PA.). The antibiotic can beeluted from said resins by using acetone:water 1:1 (v/v).

Resins other than XAD-2, and XAD-4 may be substituted. Charcoal can alsobe used. Extraction with a solvent like 1-butanol also can be used.

The eluting solvent from the resins will vary from resin to resin. Acombination of water and acetone (10:90v/v) can be used.

Purification of the antibiotic from the resin eluate can be done bychromatography on silica gel and subsequent crystallization frommethanol.

Base addition salts (e.g. metal salts, for example, sodium, calcium,magnesium and potassium) and other salts, for example, ammonium andtriethyl ammonium of antibiotic U-68,204 can be made by use of standardprocedures. These salts can be used for the same purposes as the parentantibiotic.

The following examples are illustrative of the process and product ofthe invention, but are not to be construed as limiting. All percentagesare by weight and all solvent mixture proportions are by volume unlessotherwise noted.

EXAMPLE 1

A. Fermentation

A biologically pure culture of Streptomyces thiolactonus NRRL 15439 isused to inoculate 500-ml Erlenmeyer seed flasks containing 100 ml ofsterile medium consisting of the following ingredients:

    ______________________________________                                        Bacto-Tryptone (Difco)                                                                            0.5                                                       Bacto-Yeast Extract (Difco)                                                                       0.3                                                       Deionized H.sub.2 O 100 ml                                                    Approx. pH 7.0 (6.8-7.2)                                                      ______________________________________                                    

Supplement the above with 1% glucose which has been sterilizedseparately as a 50% solution.

The seed inoculum is grown for three days at 28° C. on a New Brunswickrotary shaker operating at 250 rpm and having a 21/2 inch stroke.

Seed inoculum (5% seed), prepared as described above, is used toinoculate 500-ml Erlenmeyer flasks containing 100 l. of the followingsterile medium:

    ______________________________________                                                         g/liter                                                      ______________________________________                                        Cerelose           20                                                         Corn steep liquor  20                                                         Pharmamedia        3                                                          CaCO.sub.3         4                                                          (NH.sub.4).sub.2 SO.sub.4                                                                        3                                                          ZnSO.sub.4.7H.sub.2 O                                                                            0.03                                                       Tap water q.s. to 1 liter.                                                    ______________________________________                                    

pH is adjusted to 7.2 with KOH before sterilization. The inoculatedfermentation medium is incubated at a temperature of 28° C. for 2-3 dayson a New Brunswick rotary shaker as described above.

A typical fermentation has the following titers of antibiotic in thefermentation broth using a paper disc assay:

    ______________________________________                                               Ps. aeruginosa UC 9027                                                                      B. fragilis UC 6513                                      Fermentation                                                                           Inhibition* Bio-    Inhibition*                                                                             Bio-                                   Days pH      (mm, diameter)                                                                            units (mm, diameter)                                                                          units                                ______________________________________                                        2    7.5-8.3 24-27       1.1-2.0                                                                             23-26     1.5-1.8                              3    8.0-8.6 23-26       1.5-2.0                                                                             23-26     1.3-1.8                              ______________________________________                                         *dipped 1/2 in. paper disc                                               

A BU (biounit) is the concentration of the antibiotic which gives a 20mm zone of inhibition against the test organism.

B. Recovery

Filtered beer at harvest pH (8.5±0.2) is percolated over a bed of XAD-4(Rohm & Haas Co.) equal in volume to 10% of the beer volume. The resinbed is washed with deionized water until the column elutate is clear.The antibiotic is removed from the column by 1:1 acetone:water (V/V). Agummy solid can be obtained by stripping off the acetone, adjusting theaqueous phase to pH 3 with hydrochloric acid, extracting into methylenechloride (3× with 1/3 volumes) and stripping off the methylene chloride.At this stage the product is a brown oil.

The filtration can be accomplished using common filter aids such asDicalite, Harborlite or Celite products. The acetone may be replaced byother volatile, water-miscible solvents such as methanol, ethanol,tetrahydrofuran and acetonitrile. The amount of solvent in the aqueousstream may be varied from 1 to 80% (V/V). The pH adjustment can be madewith any convenient organic or mineral acid such as sulfuric,hydrochloric or p-toluenesulfonic. The final pH can range from 0 to 5.The CH₂ Cl₂ can be replaced with ethyl acetate, 1-butanol, chloroform orlike solvents.

A convenient alternative process consists of first extracting thefiltered beer (pH 8.5±0.2) with a water-immiscible solvent (such asethyl acetate, chloroform, methylene chloride, 1-butanol, amyl acetate)to remove lipophilic materials. The aqueous phase can then be adjustedto pH 3 with a mineral acid (such as HCl, H₂ SO₄, HClO₄) and extractedwith any of the solvents mentioned above. Antibiotic U-68,204 isisolated as a brown oil after stripping the dried (MgSO₄,Na₂ SO₄) andfiltered organic extract.

Other resins may be used such as XAD-2, XAD-7 and XE-348 (all Rohm &Haas), or charcoal. Solvents mentioned above for XAD-2 can be used forelution purposes from these resins.

Advantage may be taken of the acidic nature of Antibiotic U-68,204. Theantibiotic can be removed from filtered beer using anion exchange resinssuch as Dowex 1 (Dow Chem. Co.), IRA 938 (Rohm & Haas), DE52 (Whatman),or DEAE Sephadex (Pharmacia). These resins can be in the chloride,acetate, hydroxide or other convenient form. Elution can be accomplishedusing solutions of ammonum chloride, ammonium hydroxide, sodiumchloride, potassium acetate, pyridinium acetate, or the like. Desaltingcan be done on one of the neutral resins described above.

C. Purification

The brown oil, obtained above, is subjected to the followingpurification procedure:

The oil is suspended in 30 ml of 9:1 MeOH:H₂ O and the solution ispassed over a column of DE52 (OH⁻) (bed vol. 600 ml). After passing 500ml of deionized water over the column, a step gradient is started. Thegradient consists of sequential elution with 500 ml each of 0.05, 0.1and 0.3M NaCl solutions. Fractions of 20 ml each are collectedthroughout. Fractions 34-67 are pooled. The pool amounts to 1.5 L andassays at 8 Bu/ml (12,000 BU total).

The pool (1.5 L) is desalted by percolating it over a 500 ml bed ofXAD-4. The column is washed with 500 ml of water and eluted with 25%acetone in water (V/V). The active fractions are pooled, concentrated toan aqueous phase, and lyophilized. The brown oil amounts to 550 mg at 15BU/mg. The value at 303 nm is 10 which indicates this is about 25% pureantibiotic U-68,204. This product is also suitable for finalpurification by CCCD. This product is now loaded into a 500 tube CCCDmachine. The solvent is 6:5:4 chloroform:methanol:pH 4.2 acetate buffermolarity. The K value is 0.7 (tubes 185-220 are pooled).

This pool is acidified with 1N HCl and the phases are separated. Theupper phase is extracted twice with half-volumes of chloroform. Thecombined organic phases are dried with magnesium sulfate, filtered andconcentrated on a rotary evaporator at a temperature of 40° C. underaspirator vacuum. The residue is dissolved in 25 ml of methanol andfiltered through a 5 micron Gelman syringe filter membrane. The filtrateis concentrated on a rotary evaporator. The oily residue is dissolved in0.1N NH₄ OH and the solution is lyophilized. The yield is 73.5 mg ofslightly yellow solid antibiotic U-68,204.

The yellowish solid obtained from CCCD as described above is furthertreated as follows:

Dissolve 670 mg of the yellow solid in 20 ml of 0.1N NH₄ OH and acidifyto pH 2 using 1N HCl. Extract the milky white ppt into CH₂ Cl₂ (3× w.equal volumes). Combine the organic phases. Dry with MgSO₄ and filterthrough a medium porosity sintered glass funnel. The methylene chloridesolution is poured into 75 ml of cyclohexane and the methylene chlorideis evaporated by placing the flask in a warm-water bath. At the pointwhere cloudiness persists, the flask is removed. Crystallization beginsat room temperature and after three hours, the flask is cooled to 5° C.for 10 hrs. The white plates are collected and dried at room temperaturein vacuo. A yield of 429 mg of an essentially pure preparation ofantibiotic U-68,204 is obtained.

The product can also be purified and crystallized in a number of saltforms. In this case, the crystallization solvents are reversed becauseof the greatly increased polarity of the salt forms. The volatilesolvent should now be polar, such as water or methanol. To this shouldbe added a less polar, miscible solvent such as acetone, acetonitrile orethyl acetate.

Salts can be made of any metal from Groups 1-8 of the Periodic Chart ofthe Elements. Examples include calcium, sodium, silver, titanium andtungsten. The two alternative procedures described above can be employedfor their synthesis. In addition, salts from amines such as mono-, di-and tri-alkyl amines can be prepared also. These include dimethylaminedicyclohexylamine, benzylamine, adamantylamine, trioctylamine andcinchonidine.

Salts, acylates and ethers of antibiotic U-68,204 can be used for thesame biological purposes as antibiotic U-68,204.

The ammonium salt is prepared by elution of antibiotic U-68,204 from aDE52 column with NH₄ OH. It can also be prepared by neutralization ofsolutions containing antibiotic U-68,204 with NH₄ OH.

The sodium salt can be prepared by antibiotic U-68,204 from a DE52column with sodium chloride.

The di- and tri-trimethylsilyl (TMS) derivatives can be made for thepurpose of GC-MS analysis by heating samples containing antibioticU-68,204 with bistrimethylsilyltrifluoroacetamide (BSTFA) andchlorotrimethylsilane. The monotrifluoroacetyl derivative can beprepared for GC-MS by using trifluoroacetic anhydride as the acylationreagent.

Numerous variations on trimethylsilyl can be made. These includebutyldimethylsilyl, triphenylsilyl and butylethylmethylsilyl. They canbe added 1, 2 or 3 units per molecules of U-68,204 to give mono-, di-and tri-silylated derivatives. Acylation can be accomplished usingvarious carboxylic acids such as benzoic, trifluoracetic,heptafluorobutyric, hexadecanoic or tiglic acids. In addition, etherscan be made such as the methyl, ethyl or benzyl ether of U-68,204.Combination derivatives can be made such as the benzyl ether of theacetate, mono- or di-TMS ethers. These are all standard procedures wellknown to persons skilled in the art. ##STR1##

We claim:
 1. Antibiotic U-68,204, which is active against Gram-positivebacteria, and which in its essentially pure form has the followingcharacteristics: (a) molecular weight of 267;(b) color of crystals:white; (c) is soluble in most organic solvents, but not in water; (d) acharacteristic infrared absorption spectrum when dissolved in a mineraloil mull as shown in FIG. 1; (e) a characteristic UV spectrum as shownin FIG. 2; (f) a characteristic ¹ H NMR spectrum as shown in FIG. 3; (g)a characteristic ¹³ C-NMR spectrum as shown in FIG. 4; (h) a molecularformula C₁₃ ₁₇ NO₃ S; and, (i) a melting point of 98°-101°.
 2. Baseaddition salts of antibiotic U-68,204 as defined in claim
 1. 3. Aprocess for preparing antibitoic U-68,204 as defined in claim 1 whichcomprises cultivating Streptomyces thiolactonus, having the identifyingcharacteristics of NRRL 15439, in an aqueous nutrient medium underaerobic conditions until substantial antibiotic U-68,204 activity isimparted to said medium.
 4. A process, according to claim 3, whereinsaid aqueous nutrient medium contains a source of assimilablecarbohydrate and assimilable nitrogen.
 5. A process according to claim 3further comprising recovering antibiotic U-68,204 from a fermentationbeer as follows:(a) percolating antibiotic U-68,204 filteredfermentation beer over a non-ionic macroporous resin, and, (b) elutingantibiotic U-68,204 from said resin with a solvent for antibioticU-68,204.